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1.
J Pharm Biomed Anal ; 91: 151-9, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24463043

ABSTRACT

Oligonucleotide aptamers are selected from libraries typically comprising up to 10(15) different sequences by an iterative process of binding, separation, amplification and purification, called SELEX. During this process, the diversity of the oligonucleotide pool decreases until, presumably, only sequences with highest binding affinities towards chosen targets remain. This selection technique is time-consuming, labor-intensive and expensive. Though well posed in principles, the SELEX procedure is noise sensitive, due to amplification of unspecific-binding sequences, and it is not surprising that aptamer selection is often not successful in practice. In view of that, a follow-up of the progress of selection during its course with simple yet reliable methods is necessary. In this paper, we describe five independent assays to estimate the sequence complexity of SELEX pools including qualitative restriction fragment length polymorphism analysis, melting curve analysis, quantitative fluorescence intensity measurements of bound ssDNA, real time PCR quantification and pool dissociation constant analysis during the progress of aptamer selection against streptavidin. Properties and features of each method are discussed and compared. Pool dissociation constant analysis and sequencing serve as reference methods.


Subject(s)
Aptamers, Nucleotide/chemistry , Oligonucleotides/chemistry , SELEX Aptamer Technique/methods , Streptavidin/chemistry
2.
J Med Chem ; 52(12): 3774-83, 2009 Jun 25.
Article in English | MEDLINE | ID: mdl-19469547

ABSTRACT

Most of human gastrointestinal stromal tumors (GIST) are driven by activating mutations in the proto-oncogene KIT, a tyrosine kinase receptor. Clinical treatment with imatinib targets the kinase domain of KIT, but tumor regrowth occurs as a result of the development of resistant mutations in the kinase active site. An alternative small-molecule approach to GIST therapy is described, in which the KIT gene is directly targeted, and thus, kinase resistance may be circumvented. A naphthalene diimide derivative has been used to demonstrate the concept of dual quadruplex targeting. This compound strongly stabilizes both telomeric quadruplex DNA and quadruplex sites in the KIT promoter in vitro. It is shown here that the compound is a potent inducer of growth arrest in a patient-derived GIST cell line at a concentration (approximately 1 microM) that also results in effective inhibition of telomerase activity and almost complete suppression of KIT mRNA and KIT protein expression. Molecular modeling studies with a telomeric quadruplex have been used to rationalize aspects of the experimental quadruplex melting data.


Subject(s)
Gastrointestinal Stromal Tumors/drug therapy , Phenanthrolines/pharmacology , Proto-Oncogene Proteins c-kit/drug effects , Base Sequence , Cell Line, Tumor , Cell Proliferation/drug effects , Computer Simulation , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Gastrointestinal Stromal Tumors/genetics , Humans , Imides , Models, Chemical , Models, Molecular , Molecular Sequence Data , Molecular Structure , Molecular Weight , Naphthalenes , Phenanthrolines/chemistry , Proto-Oncogene Mas , Proto-Oncogene Proteins c-kit/genetics , RNA, Messenger/drug effects , RNA, Messenger/genetics
3.
J Agric Food Chem ; 56(18): 8720-7, 2008 Sep 24.
Article in English | MEDLINE | ID: mdl-18722465

ABSTRACT

This paper reports a series of ab initio, density functional theory (DFT), and semiempirical molecular orbital (MO) calculations concerning the reaction between the ultimate carcinogen of acrylamide and guanine. Acrylamide--a product of the Maillard reaction--is present in a variety of fried and oven-cooked food. After intake, it is epoxidized by cytochrome P450 2E1 to yield the ultimate carcinogen--glycidamide. Effects of solvation were considered using the Langevin dipoles (LD) model of Florian and Warshel and the solvent reaction field (SCRF) model of Tomasi and co-workers. In silico activation free energies are in very good agreement with the experimental value of 22.8 kcal/mol. This agreement presents strong evidence in favor of the validity of the proposed S N2 reaction mechanism and points to the applicability of quantum chemical methods to studies of reactions associated with carcinogenesis. In addition, insignificant stereoselectivity of the studied reaction was predicted. Finally, the competing reaction of glycidamide with adenine was simulated, and the experimentally observed regioselectivity was successfully reproduced.


Subject(s)
Acrylamide/toxicity , Carcinogens/toxicity , Acrylamide/chemistry , Adenine/chemistry , Carcinogenicity Tests , Carcinogens/chemistry , Epoxy Compounds/chemistry , Food Analysis , Hot Temperature , Maillard Reaction , Mathematics , Thermodynamics
4.
Protein Expr Purif ; 53(1): 104-11, 2007 May.
Article in English | MEDLINE | ID: mdl-17223361

ABSTRACT

A member of the cysteine protease inhibitor clitocypin gene family from basidiomycete Clitocybe nebularis was expressed in Escherichia coli. Following careful optimization of the expression procedure the active inhibitor was purified from inclusion bodies and its properties examined and compared to those of the natural clitocypin. The CD spectrum of recombinant clitocypin was similar to that of natural clitocypin, indicating that protein was properly refolded during purification. In spite of some differences in primary structure, structural, functional and immunological equivalence was established. Kinetic analyses of the natural and recombinant clitocypins were performed. Both clitocypins inhibited a range of cysteine proteases to a similar extent, and demonstrated an unusually broad inhibitory spectrum, including distantly related proteases, such as papain and legumain, belonging to different protease families. The homogenous, biologically active recombinant clitocypin is obtained at levels adequate for further structure-function studies.


Subject(s)
Basidiomycota/enzymology , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Amino Acid Sequence , Antibodies/immunology , Chromatography, Ion Exchange , Circular Dichroism , Cloning, Molecular , Cysteine Proteinase Inhibitors/genetics , Cysteine Proteinase Inhibitors/isolation & purification , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/genetics , Fungal Proteins/immunology , Fungal Proteins/isolation & purification , Hot Temperature , Immunoblotting , Inclusion Bodies/chemistry , Isoelectric Focusing , Kinetics , Molecular Sequence Data , Plasmids , Protein Binding , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Spectrometry, Mass, Electrospray Ionization
5.
Article in English | MEDLINE | ID: mdl-16511249

ABSTRACT

Clitocypin is a cysteine protease inhibitor from the mushroom Clitocybe nebularis. The protein has been purified from natural sources and crystallized in a variety of non-isomorphous forms belonging to monoclinic and triclinic space groups. A diffraction data set to 1.55 A resolution was obtained from a crystal belonging to space group P2, with unit-cell parameters a = 38.326, b = 33.597, c = 55.568 A, beta = 104 degrees. An inability to achieve isomorphism forced the use of MAD and SAD phasing methods. Phasing is in progress.


Subject(s)
Agaricales/enzymology , Cysteine Proteinase Inhibitors/chemistry , Fungal Proteins/chemistry , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/isolation & purification , Fungal Proteins/isolation & purification
6.
Biochem Biophys Res Commun ; 324(2): 576-8, 2004 Nov 12.
Article in English | MEDLINE | ID: mdl-15474465

ABSTRACT

The molecular mass of clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, has been determined by analytical ultracentrifugation and gel exclusion chromatography. The result is in agreement with the formula mass of 16.8 kDa, demonstrating that the inhibitor is a monomer in aqueous solution. This enables the kinetics of unfolding and refolding to be interpreted in terms of folding in a kinetically two state, highly cooperative transition from the thermally unfolded state.


Subject(s)
Cysteine Proteinase Inhibitors/pharmacology , Fungal Proteins/physiology , Chromatography, Gel , Dimerization , Enzyme Inhibitors/pharmacology , Fungal Proteins/chemistry , Hydrogen-Ion Concentration , Kinetics , Protein Folding , Protein Structure, Tertiary , Temperature , Time Factors , Ultracentrifugation
7.
FEBS Lett ; 539(1-3): 120-4, 2003 Mar 27.
Article in English | MEDLINE | ID: mdl-12650938

ABSTRACT

Equistatin is a protein composed of three thyroglobulin type-1 domains. It inhibits papain-like cysteine proteinases and the aspartic proteinase, cathepsin D. To determine the structural basis for this inhibition we cloned and expressed the separated domains (eq d-1, eq d-2, eq d-3) in Pichia pastoris. Kinetic constants for the interaction of eq d-1 with papain and that of eq d-2 with cathepsin D are of similar order (subnanomolar) and are comparable to the constants obtained for full-length equistatin. The target proteinase for the third domain remains unknown. Thus, we demonstrate here that thyroglobulin type-1 motifs per se are able to support specific structural features that enable them to inhibit proteases from different classes. The overall conformation of three domains in equistatin is such that the interaction of domains 1 or 2 with their respective target enzymes is not hindered sterically by either domain. In addition, we show that the interaction of eq d-2 with cathepsin D results in conformational changes, which is not the case for the eq d-1/papain interaction.


Subject(s)
Proteins/chemistry , Cathepsin D/antagonists & inhibitors , Circular Dichroism , Cloning, Molecular , Kinetics , Papain/metabolism , Pichia , Protein Structure, Tertiary , Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Thyroglobulin/chemistry
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